Sialic Acids (Sias) are molecules attached to the outer ends of sugar chains (glycans) on cell surface and secreted molecules of vertebrates. The long-term goal of this grant has been to unravel mechanisms regulating various kinds of Sia attachments and structural modifications, and to elucidate their biological and pathological roles. All attention is now focused on the human-specific genetic loss of the common mammalian Sia N- glycolylneuraminic acid (Neu5Gc), which differs by a single oxygen atom from its precursor N-acetylneuraminic acid (Neu5Ac), which is enriched in humans. We have found that human cells can take up and metabolically incorporate exogenous free or bound Neu5Gc in significant amounts, and that this is relevant to human dietary intake of Neu5Gc, allowing incorporation into certain human cell types in vivo, as well Neu5Gc incorporation into biotherapeutic products in vitro. Our general hypothesis is that the loss of Neu5Gc in humans has broad implications, ranging from basic biochemical and cell biological issues involving Neu5Gc uptake and incorporation into some cell types, to the origins, diversity and significance of anti-Neu5Gc antibodies in humans, and to the need to develop methods to eliminate Neu5Gc from human cells and tissues. Besides biochemical, cell biological and epidemiological studies of human cells and humans, we will use mice with a human-like genetic defect in Neu5Gc production. We will study the metabolism and fate of Neu5Gc in human and animal cells and in animal models, asking if humans are uniquely different. Since the mice with a human- like defect do not easily accumulate dietary Neu5Gc, we hypothesize that mammalian cells have an as yet unknown mechanism to turnover Neu5Gc (and/or the N-glycolyl group), and that this mechanism may be defective in humans (as >2 million years have elapsed since we lost the ability to synthesize Neu5Gc). We will follow the fate of double-labeled Neu5Gc in human animal cells, and propose biochemical, bioinformatics and genetic approaches to discover the putative gene (Gene X) involved in turnover of Neu5Gc in rodent cells. At the organismal level, we will follow the fate of bound and free Neu5Gc in the mouse GI tract, addressing host and microbial factors affecting its metabolic incorporation. Meanwhile, we will explore mechanisms by which adult humans develop a diverse and variable range of anti-Neu5Gc antibodies, testing multiple hypotheses regarding this issue, and also asking whether pre- or postnatal exposure to Neu5Gc impacts antibody production. We will also study pathological consequences of in vivo interactions between human anti-Neu5Gc antibodies and Neu5Gc-containing glycans, with a particular focus on the exacerbation of atherosclerosis. If time allows, we will ask whether the Neu5Gc found on biotherapeutic agents has any negative impact. Finally, assuming we confirm the importance of Neu5Gc and anti-Neu5Gc antibodies for human disease and/or biotherapeutic agents, we will pursue promising preliminary data, to develop simple and non-toxic metabolic manipulations that eliminate Neu5Gc from cells in culture, and potentially from the human body.